Targeted cleavage of RNA using eukaryotic ribonuclease P and external guide sequence

Abstract

It has been discovered that any RNA can be targeted for cleavage by RNAase P from eukaryotic cells, for example, human RNAase P, using a suitably designed oligoribonucleotide ("external guide sequence", or EGS) to form a hybrid with the target RNA, thereby creating a substrate for cleavage by RNAase P in vitro. The EGS hydrogen bonds to the targeted RNA to form a partial tRNA like structure including the aminoacyl acceptor stem, the T stem and loop, and part of the D stem. The most efficient EGS with human RNAase P is the EGS in which the anticodon stem and loop was deleted. Modifications can also be made within the T-loop. Methods are also disclosed to randomly select and to express a suitable EGS in vivo to make a selected RNA a target for cleavage by the host cell RNAase P, thus preventing expression of the function of the target RNA. The methods and compositions should be useful to prevent the expression of disease-causing genes in vivo.

We claim: 
     
       1. A composition for targeting an RNA substrate for cleavage by eukaryotic RNAase P comprising a recombinant external guide sequence molecule including   a cleavage targeting sequence, and   a nucleotide sequence complementary to the substrate,   wherein the external guide sequence and complementary nucleotide sequence base pairs to the substrate to form a hybrid structure having secondary structure resembling a precursor tRNA under conditions promoting cleavage by the RNAase P of the substrate at the nucleotide at the 5' end of the base-paired region, wherein the hybrid structure comprises a dihydrouridine stem, T stem and loop, aminoacyl acceptor stem, and either an anticodon stem and loop or variable loop.   
     
     
       2. The composition of claim 1 wherein the external guide sequence is complementary to a target sequence at least eleven nucleotides in length and comprising seven nucleotides base pairing with the external guide sequence to form a structure similar to the aminoacyl acceptor stem of a precursor tRNA, followed by two nucleotides not base pairing with the external guide sequence, followed by four nucleotides base pairing with the external guide sequence to form a structure similar to the dihydrouridine stem of a precursor tRNA. 
     
     
       3. The composition of claim 1 wherein the RNAase P is selected from the group consisting of yeast and mammalian RNAase P. 
     
     
       4. The composition of claim 1 wherein the variable loop comprises at least one nucleotide base pair not hydrogen bonding to the target sequence which is located between the external guide sequence forming the T loop and stem and the dihydrouridine stem. 
     
     
       5. The composition of claim 2 wherein the external guide sequence comprises an anticodon stem and loop or portions thereof. 
     
     
       6. The composition of claim 2 wherein the external guide sequence comprises a variable stem and loop or portions thereof. 
     
     
       7. The composition of claim 1 wherein the external guide sequence is modified in a region selected from the group consisting of the T loop, anticodon stem and loop, and variable stem and loop and is in a form to limit degradation of the external guide sequence. 
     
     
       8. The composition of claim 1 wherein the external guide sequence is modified in a region selected from the group consisting of the T loop, anticodon stem and loop, and variable stem and loop so that it has a nucleotide sequence that is different from a naturally occuring tRNA. 
     
     
       9. The composition of claim 1 wherein the RNAase P is targeted to sequences such that cleavage of the sequences by the RNAase P results in inactivation of RNA selected from the group consisting of RNA complementary to oncogenes, RNA complementary to tumor suppressor genes, RNA complementary to viral genes and RNA viral genes, and cellular mRNAs which encode proteins selected from the group consisting of enzymes, hormones, cofactors, antibodies, and growth factors. 
     
     
       10. The composition of claim 1 further comprising a pharmaceutical carrier selected from the group consisting of carriers suitable for topical, subcutaneous, parental, and enteral administration. 
     
     
       11. The composition of claim 1 wherein the external guide sequence is in a vector for introducing the external guide sequence into a cell containing the RNA targeted for cleavage. 
     
     
       12. The composition of claim 11 wherein the vector is a retroviral vector. 
     
     
       13. A method for specifically cleaving an RNA substrate comprising providing in combination with RNAase P, an external guide sequence including a cleavage targeting sequence, and   a nucleotide sequence complementary to the substrate,   wherein the external guide sequence base pairs to the substrate to form a hybrid structure having secondary structure resembling a precursor tRNA under conditions promoting cleavage by the RNAase P of the substrate at the nucleotide at the 5' end of the base-paired region, wherein the hybrid structure comprises a dihydrouridine stem, T stem and loop, aminoacyl acceptor stem, and either an anticodon stem and loop or variable loop, and   administering the external guide sequence to cells containing the substrate which forms a hybrid structure with the external guide sequence and is cleaved by RNAase P.   
     
     
       14. The method of claim 13 wherein the external guide sequence is complementary to a target sequence at least eleven nucleotides in length and comprising seven nucleotides base pairing with the external guide sequence to form a structure similar to the aminoacyl acceptor stem of a precursor tRNA, followed by two nucleotides not base pairing with the external guide sequence, followed by four nucleotides base pairing with the external guide sequence to form a structure similar to the dihydrouridine stem of a precursor tRNA. 
     
     
       15. The method of claim 13 wherein the RNAase P is targeted to sequences such that cleavage of the sequences by the RNAase P results in inactivation of RNA selected from the group consisting of RNA complementary to oncogenes. RNA complementary to tumor suppressor genes, RNA complementary to viral genes and RNA viral genes, and cellular mRNAs which encode proteins selected from the group consisting of enzymes, hormones, cofactors, antibodies, and growth factors. 
     
     
       16. The method of claim 13 further comprising providing the external guide sequence in a vector for introducing the external guide sequence into a cell containing the RNA targeted for cleavage. 
     
     
       17. The composition of claim 1 wherein the external guide sequence is selected by randomizing a section of the starting external guide sequence;   selecting for a subpopulation of the randomized sequences for their ability to be cleaved efficiently by RNAase P;   amplifying those sequences cleaving more efficiently than the starting external guide sequence; and   repeating the selection and amplification steps.